IFNAR2/IFN complex

ABSTRACT

The in vivo effect of Type I interferon (IFN) can be prolonged by administering the interferon in the form of a complex with an IFN binding chain of the human interferon α/β receptor (IFNAR). Such a complex also improves the stability of the IFN and enhances the potency of the IFN. The complex may be a non-covalent complex or one in which the IFN and the IFNAR are bound by a covalent bond or a peptide. When bound by a peptide bond in the form of a fusion protein, the IFN may be separated from the IFNAR by means of a peptide linker. Such a fusion protein may be produced by recombinant DNA technology. Storing IFN in the form of such a complex improves the storage life of the IFN and permits storage under milder conditions than would otherwise be possible.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. provisionalapplication Ser. No. 60/068,295, filed Dec. 19, 1997, the entirecontents of which are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to a Type I interferon complex, composedof the polypeptide sequence of the human interferon α/β receptor(IFNAR2) extracellular domain and a Type I interferon (IFNα, IFNβ, andIFNω). Such a complex improves the stability, enhances the potency, andprolongs the pharmacokinetics in vivo of free IFN for anti-viral,anti-cancer and immune modulating activity. More particularly, thecomplex is a fusion protein, or a covalent complex, or a non-covalentcomplex containing the polypeptide sequence of the entire extracellulardomain of IFNAR2, or any interferon-binding subfraction thereof,complexed to a Type I interferon (IFNα, IFNβ, IFNω), or any biologicallyactive subfraction thereof.

BACKGROUND OF INVENTION

Interferons are classified either as the leukocyte and fibroblastderived Type I interferons, or as the mitogen induced or “immune” TypeII interferons (Pestka et al, 1987). Through analysis of sequenceidentities and common biological activities, Type I interferons includeinterferon alpha (IFNα), interferon beta (IFNβ) and interferon omega(IFNω), while Type II interferon includes interferon gamma (IFNγ). TheIFNα, IFNβ and IFNω genes are clustered on the short arm of chromosome 9(Lengyl, 1982). There are at least 25 non-allelic IFNα genes, 6non-allelic IFNω genes and a single IFNβ gene. All are believed to haveevolved from a single common ancestral gene. Within species, IFNα genesshare at least 80% sequence identity with each other. The IFNβ geneshares approximately 50% sequence identity with IFNα; and the IFNω geneshares 70% homology with IFNα (Weissman et al, 1986; Dron et al, 1992).IFNα has a molecular weight range of 17-23 kDa (165-166 amino acids),IFNβ, ˜23 kDa (166 amino acids) and IFNω, ˜24 kDa (172 amino acids).

Type I interferons are pleiotropic cytokines having activity in hostdefense against viral and parasitic infections, as anti-cancer cytokinesand as immune modulators (Baron et al, 1994; Baron et al, 1991). Type Iinterferon physiological responses include anti-proliferative activityon normal and transformed cells; stimulation of cytotoxic activity inlymphocytes, natural killer cells and phagocytic cells; modulation ofcellular differentiation; stimulation of expression of class I MHCantigens; inhibition of class II MHC; and modulation of a variety ofcell surface receptors. Under normal physiological conditions, IFNα andIFNβ (IFNα/β) are secreted constitutively by most human cells at lowlevels with expression being up-regulated by addition of a variety ofinducers, including infectious agents (viruses, bacteria, mycoplasma andprotozoa), dsRNA, and cytokines (M-CSF, IL-1α, IL-2, TNFα). The actionsof Type I interferon in vivo can be monitored using the surrogatemarkers, neopterin, 2′, 5′ oligoadenylate synthetase, and β2microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; Salmon et al,1996).

Type I interferons IFNα/β/ω) act through a cell surface receptor complexto induce specific biologic effects, such as anti-viral, anti-tumor, andimmune modulatory activity. The Type I IFN receptor (IFNAR) is ahetero-multimeric receptor complex composed of at least two differentpolypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993;Platanias et al, 1993). The genes for these chains are found onchromosome 21, and their proteins are expressed on the surface of mostcells (Tan et al, 1973). The receptor chains were originally designatedalpha and beta because of their ability to be recognized by themonoclonal antibodies IFNαR3 and IFNaRβ1, respectively. Most recently,these have been renamed IFNAR1 for the alpha subunit and IFNAR2 for thebeta subunit. In most cells, IFNAR1 (alpha chain, Uze subunit) (Uze etal, 1990) has a molecular weight of 100-130 kDa, while IFNAR2 (betachain, B_(L), IFNα/βR) has a molecular weight of 100 kDa. In certaincell types (monocytic cell lines and normal bone marrow cells) analternate receptor complex has been identified, where the IFNAR2 subunit(β_(S)) is expressed as a truncated receptor with a molecular weight of51 kDa. The IFNAR1 and IFNAR2 β_(S) and β_(L) subunits have been cloned(Novick et al, 1994; Domanski et al, 1995). The IFNAR2 β_(S) and β_(L)subunits have identical extracellular and transmembrane domains;however, in the cytoplasmic domain they only share identity in the first15 amino acids. The IFNAR2 subunit alone is able to bind IFNα/β, whilethe IFNAR1 subunit is unable to bind IFNα/β. When the human IFNAR1receptor subunit alone was transfected into murine L-929 fibroblasts, nohuman IFNαs except IFNα8/IFNαB were able to bind to the cells (Uze etal, 1990). The human IFNAR2 subunit, transfected into L cells in theabsence of the human IFNAR1 subunit, bind human IFNα2, binding with a Kdof approximately 0.45 nM. When human IFNAR2 subunits were transfected inthe presence of the human IFNAR1 subunit, high affinity binding could beshown with a Kd of 0.026-0.114 nM (Novick et al, 1994; Domanski et al,1995). It is estimated that from 500-20,000 high affinity and2,000-100,000 low affinity IFN binding sites exist on most cells.Although the IFNAR1/2 complex (α/β_(s) or α/β_(L)) subunits bind IFNαwith high affinity, only the α/β_(L) pair appears to be a functionalsignaling receptor.

Transfection of the IFNAR1 and the IFNAR2 β_(L) subunits into mouseL-929 cells, followed by incubation with IFNα2, induces an anti-viralstate, initiates intracellular protein phosphorylation, and causes theactivation of intracellular kinases (Jak1 and Tyk2) and transcriptionfactors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995).In a corresponding experiment, transfection of the IFNAR2 β_(S) subunitwas unable to initiate a similar response. Thus, the IFNAR2 β_(L)subunit is required for functional activity (anti-viral response) withmaximal induction occurring in association with the IFNAR1 subunit.

In addition to membrane bound cell surface IFNAR forms, a soluble IFNARhas been identified in both human urine and serum (Novick et al, 1994;Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). Thesoluble IFNAR isolated from serum has an apparent molecular weight of 55kDa on SDS-PAGE, while the soluble IFNAR from urine has an apparentmolecular weight of 40-45 kDa (p40). Transcripts for the soluble p40IFNAR2 are present at the mRNA level and encompass almost the entireextracellular domain of the IFNAR2 subunit with two new amino acids atthe carboxy terminal end. There are five potential glycosylation siteson the soluble IFNAR2 receptor. The soluble p40 IFNAR2 has been shown tobind IFNα2 and IFNβ and to inhibit in vitro the anti-viral activity of amixture of IFNα species (“leukocyte IFN”) and individual Type I IFNs(Novick et al, 1995). A recombinant IFNAR2 subunit Ig fusion protein wasshown to inhibit the binding of a variety of Type I IFN species (IFNαA,IFNαB, IFNαD, IFNβ, IFNα Con1 and IFNω) to Daudi cells and α/β_(S)subunit double transfected COS cells.

Type I IFN signaling pathways have recently been identified (Plataniaset al, 1996; Yan et al, 1996; Qureshi et al, 1996; Duncan et al, 1996;Sharf et al, 1995; Yang et al, 1996). Initial events leading tosignaling are thought to occur by the binding of IFNα/β/ω to the IFNAR2subunit, followed by the IFNAR1 subunit associating to form an IFNAR1/2complex (Platanias et al, 1994). The binding of IFNα/β/ω to the IFNAR1/2complex results in the activation of two Janus kinases (Jak1 and Tyk2)which are believed to phosphorylate specific tyrosines on the IFNAR1 andIFNAR2 subunits. Once these subunits are phosphorylated, STAT molecules(STAT 1, 2 and 3) are phosphorylated, which results in dimerization ofSTAT transcription complexes followed by nuclear localization of thetranscription complex and the activation of specific IFN induciblegenes.

The pharmacokinetics and pharmacodynamics of Type I IFNs have beenassessed in humans (Alan et al, 1997; Fierlbeck et al, 1996; Salmon etal, 1996). The clearance of IFNβ is fairly rapid with thebioavailability of IFNβ lower than expected for most cytokines. Althoughthe pharmacodynamics of IFNβ have been assessed in humans, no clearcorrelation has been established between the bioavailability of IFNβ andclinical efficacy. In normal healthy human volunteers, administration ofa single intravenous (iv) bolus dose (6 MIU) of recombinant CHO derivedIFNβ resulted in a rapid distribution phase of 5 minutes and a terminalhalf-life of ˜5 hours (Alam et al, 1997). Following subcutaneous (sc) orintramuscular (im) administration of IFNβ, serum levels are flat withonly ˜15% of the dose systemically available. The pharmacodynamics ofIFNβ following iv, im or sc administration (as measured by changes in2′5′-oligoadenylate synthetase (2′,5′-AS) activity in PBMCs) wereelevated within the first 24 hours and slowly decreased to baselinelevels over the next 4 days. The magnitude and duration of the biologiceffect was the same regardless of the route of administration.

The pharmacokinetics (PK) and pharmacodynamics (PD) of IFNβ manufacturedby two different companies (REBIF®-Serono and AVONEX®-Biogen) has beenexamined following the im injection of a single dose of 6 MIU ofrecombinant IFNβ (Salmon, 1996). Serum concentration of IFNβ and theIFNβ surrogate marker, neopterin, were monitored over time. Both IFNβpreparations exhibited similar PK profiles with peak serum levels ofIFNβ achieved by ˜12-15 hours, although REBIF® gave lower maximumlevels. The IFNβ levels remained elevated for both REBIF® and AVONEX®for at least the first 36 hours post im injection and then dropped toslightly above baseline by 48 hours. Levels of neopterin exhibited avery similar profile between REBIF® and AVONEX® with maximal neopterinlevels achieved at ˜44-50 hours post-injection, remaining elevated until72 hours post-injection and then dropping to baseline gradually by 144hours.

A multiple dose pharmacodynamic study of IFNβ has been conducted inhuman melanoma patients (Fierlbeck et al, 1996) with IFNβ beingadministrated by sc route, three times per week at 3 MIU/dose over asix-month period. The pharmacodynamic markers, 2′, 5′-AS synthetase,β2-microglobulin, neopterin, and NK cell activation peaked by the secondinjection (day 4) and dropped off by 28 days, remaining only slightlyelevated out to six months.

In summary, the clearance of Type I interferons in humans is rapid. Along-acting interferon preparation would likely result in an improvementin clinical benefit.

SUMMARY OF THE INVENTION

It has now been found that a Type I interferon complex, composed ofsoluble IFNAR complexed with Type I interferons (IFN), exhibits improvedstability, enhanced potency, and elongated pharmacokinetics in vivocompared with free IFN for anti-viral, anti-cancer and immune modulatingactivity.

The present invention thus provides a Type I interferon (IFN) complex,composed of the polypeptide sequence of a human interferon α/β receptor(IFNAR) subunit extracellular domain and Type I interferons, whichexhibits improved stability, enhanced potency, and/or prolongedpharmacokinetics in vivo compared to free IFN for anti-viral,anti-cancer and immune modulating activity. Preferably, the complex isof the IFNAR2 subunit extracellular domain with any Type I interferon orthe IFNAR1 subunit with IFNα.

More specifically, the complex is a fusion protein, or a covalentcomplex, or a non-covalent complex containing the polypeptide sequenceof the entire extracellular domain of IFNAR, preferably IFNAR2, or anyinterferon-binding subfraction thereof, complexed to IFNα or IFNβ orIFNω, or any biologically active subfraction thereof.

IFNAR is intended to comprehend any of the known extracellular IFNARreceptors as defined above, as well as any active fragments thereof.IFNAR can be optionally fused to another protein, for example, animmunoglobulin such as IgG. IFN, IFNα, IFNβ, and IFNω are intended asone of the more than 20 Type I interferons identified to date, or anyother Type I interferon identified in the future.

In one embodiment of the present invention, the complex is composed ofIFNα or IFNβ, covalently linked to IFNAR2 via chemical linkage.

A further embodiment comprises a complex composed of IFNα or IFNβ,non-covalently complexed to IFNAR2. This further embodiment alsoincludes a composition containing a Type I IFN and IFNAR2 in any ratio.A formulation of Type I IFN with an excess of IFNAR2 as defined above isalso included in the definition of “complex” of the present application.The two components may also be administered separately so as to form thecomplex in vivo. Thus, in a further embodiment, the complex is a mixtureof IFNAR2 and IFN, obtained by simultaneous or subsequentco-administration of IFNα or IFNβ and soluble IFNAR2. Furthermore, theIFNAR can be administered without any concomitant administration of IFN,so that the complex may be formed in vivo with endogenous circulatingIFN, thereby potentiating the effects of the endogenous IFN.

As a particular embodiment, the complex is composed of IFNα or IFNβ orIFNω fused to IFNAR2 as a recombinant fusion protein, where the IFN andthe IFNAR2 moieties are optionally fused via a flexible peptide linkermolecule. This peptide linker may or may not be cleavable in vivo.

The invention further relates to DNA encoding such fusion proteins,vectors containing such DNA, host cells transformed with such vectors insuch a manner as to express the fusion proteins and methods ofproduction of such fusion proteins by culturing such host cells andisolating the fusion proteins expressed thereby.

A further aspect of the present invention are the methods of use of thecomplexes of the present invention for prolonging the in vivo effect ofIFN, which is useful in the treatment of any disease or condition whichis treatable by IFN.

Another aspect of the present invention relates to the use of IFNAR as astabilizer in formulations of IFN. Free IFNβ has a tendency tooligomerize. This is prevented once it is complexed to IFNAR,particularly IFNAR2. Present day formulations of recombinant IFNβ musthave an acidic pH, which may cause some localized irritation whenadministered. Non-acidic compositions can be formulated if IFNAR is usedas a stabilizer.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in conjunction with thefollowing drawings, in which:

FIG. 1 is a graph showing dose-dependent anti-viral activity of sIFNAR2or IFNAR2Ig (composed of the extracellular domain of hIFNAR2 fused tohuman IgG1 hinge, CH2 and CH3 domains) in the presence of IFNβ.

FIG. 2 is a graph showing anti-viral activity of sIFNAR2 and IFNβ at asub-optimal dose of IFNβ. Synergistic anti-viral activity of IFNβ andsIFNAR2 following one hour preincubation is revealed at an intermediateIFNβ dose.

FIG. 3 is a graph showing anti-viral activity of sIFNAR2 and asub-effective dose of IFNβ (0.95 IU/ml) as a function of preincubationtime. WISH cells were exposed to IFNβ (0.95 IU/ml sub-effective dose)and sIFNAR2, following a preincubation at the indicated times.Synergistic anti-viral activity is observed only after 4 hours ofpreincubation. Anti-viral activity was measured by MTT conversion 48hours after VSV challenge. At sub-therapeutic levels of IFNβ, formationof the IFNAR2/IFN complex results in enhanced anti-viral activity.

FIG. 4 is a graph showing enhanced anti-viral activity of human IFNβfollowing preincubation with sIFNAR2.

FIG. 5 is a graph showing that enhanced anti-viral activity of humanIFNβ associated with sIFNAR is specific for IFNAR2 but not otherproteins.

FIG. 6 is a graph showing the pharmacokinetic comparison of human IFNβand IFNβ/IFNAR2 complex in mice as determined by ELISA.

FIG. 7 is a graph showing the pharmacokinetic comparison of humanIFNβand IFNβ/IFNAR2 complex in mice as determined by bioassay.

FIG. 8 is a graph showing the pharmacokinetic comparison of human IFNαand IFNα/IFNAR2 complex in mice as determined by ELISA.

FIG. 9 is a graph showing the pharmacokinetic comparison of human IFNαand IFNα/IFNAR2 complex in mice as determined by bioassay.

FIG. 10 shows the amino acid sequence of the n=2 IFNAR2/IFNβ fusionprotein (SEQ ID NO:14). The (GGGGS)₂ (residues 240-249 of SEQ ID NO:14)linker is underlined.

FIG. 11 is a gel showing the restriction enzyme analysis ofpCMV-IFNAR2/IFNβ; 10% PAG, Lanes 3-7: BamHI/XhoI digests; Lane 2:SmaI/XhoI digest; Lane 1: pBR322 DNA-MspI digest marker; Lane 2:pCMV-IFNAR2-IFNβ, 0GS; Lane 3: 1GS; Lane 4: 2GS; Lane 5: 3GS; Lane 6:4GS; Lane 7: 5GS; Lane 8: φX174 RFDNA HaeIII digest marker.

FIG. 12 is the restriction endonuclease map of the IFNAR2/IFNβexpression vector.

FIG. 13 is the Western blot analysis of IFNAR2/IFNβ fusion protein. Lane1, IFNAR2/IFNβ construct containing no linker; lane 2, IFNAR2/IFNβconstruct containing one Gly₄Ser (SEQ ID NO:1) linker; lane 3,IFNAR2/IFNβ construct containing two Gly₄Ser (SEQ ID NO:1) linkers; lane4, IFNAR/IFNβ construct containing three Gly₄Ser (SEQ ID NO:1) linkers;lane 5, IFNAR/IFNβ construct containing four Gly₄Ser (SEQ ID NO:1)linkers; lane 6, IFNAR/IFNβ construct containing five Gly₄Ser (SEQ IDNO:1) linkers.

FIG. 14 is a graph showing anti-viral activity of Interfusion moleculesexpressed in CHO cells supernatants and normalized to IFNβ standardactivity.

FIGS. 15A-B are graphs showing the pharmacokinetics of IFNα administeredafter intravenous injection of IFNAR2. IFNβ was injected either alone,as an IFNAR complex, or immediately following a separate I.V. injectionof sIFNAR2. The serum half life was assessed at the indicated times postinjection by IFNβ specific ELISA (FIG. 15A) and by bioactivity in theWISH antiviral assay (FIG. 15B).

FIG. 16 is a graph showing the protective effect in terms of percentcytotoxicity of various doses of a complex of “Universal” IFN (humanIFNαA/D) in a complex with sIFNAR2, as compared to administration ofUniversal IFN alone or a control.

FIG. 17 is a graph showing the serum concentration of IFNβ as a functionof time after a single bolus intravenous injection of either InterfusionGS5 or hIFNβ alone.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The IFNAR/IFN complex of the present invention and the enablingtechnology required to produce this complex is described in detailhereinbelow. For most of the results, IFNβ has been chosen as anon-limiting example.

Fusion Protein. The C-terminal end of IFNAR2, or any interferon-bindingsubsequence thereof, has been fused to the N-terminal end of IFNβ, orbiologically active fragments thereof, as this requires the shortestdistance to be bridged between the two molecules. The reverse constructscan also be prepared, where the C-terminal end of IFNβ, or fragmentsthereof, are fused to the N-terminal end of IFNAR2, or subsequencesthereof.

From molecular models of the IFNAR2/IFNβ complex, the estimated distancebetween the constrained C-terminal extracellular domain of IFNAR2 andthe N-terminal of IFNβ in the active complex model is ˜80 Angstroms. Inorder to engineer an IFNAR2/IFNβ complex which allows the active complexto be maintained, a flexible peptide linker, for example,Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO:1) repeats, may be employed.Alternatively, the linker may be flexible and a target for proteolyticcleavage by serum, membrane bound, and/or cellular proteases. As examplefor a serum protease cleavage site the engineered IFNAR2/IFNβ complexfusion may have a Factor Xa cleavage site. Factor Xa cleaves prothrombinat two locations Arg273 and Arg322, and it has a tetrapeptiderecognition signal Ile-Glu-Glu-Arg (SEQ ID NO:2) (Nagai et al, 1984).Factor Xa itself is generated from both intrinsic and extrinsic pathwaysby a variety of activators, including tissue factor, which is releasedby vascular endothelial cells, macrophages and neutrophils.

Factor Xa can act on a sIFNAR2/IFN fusion protein containing a Factor Xarecognition sequence in the linker domain and release the IFNAR2/IFNcomplex such that the complex can function as a non-covalent complex.

Alternatively, the IFNAR2/IFNβ complex fusion may have a cell membraneprotease (e.g., hepsin) cleavage site. Hepsin is a 51 kDa membrane boundserine protease zymogen expressed in high levels in liver tissue but isalso found in kidney, pancreas, lung, thyroid, pituitary and testis. Onesequence known to be cleaved by hepsin is the Arg152-Ile153 peptide bondin Factor VII. Hepsin has been implicated in the formation of thrombinon tumor cells (Kazam et al, 1995).

Hepsin can act on a sIFNAR2/IFN fusion protein containing a hepsinrecognition sequence in the linker domain and release the IFNAR2/IFNcomplex such that the complex can function in a non-covalent fashion.

Alternatively, the IFNAR2/IFNα fusion complex may have an intracellularprotease cleavage site. A variety of proteases are released by necrosingand apoptosing cells. Included in these are caspases (Interleukin 1beta-converting enzyme-like proteases), metallo proteinases, lysosomalproteases (e.g., cathepsin B) and elastase. Elastase is released bygranulocytes during disease states (e.g., sepsis) and has a broadspecificity regarding amino acid cleavage sequence, akin to that oftrypsin (Ertel et al, 1994; Szilagyi et al, 1995).

Intracellular proteases can act on a sIFNAR2/IFN fusion proteincontaining an intracellular protease recognition sequence in the linkerdomain and release the IFNAR2/IFN complex such that the complex canfunction as a non-covalent complex.

Several examples of fusion protein constructs in which the C-terminal ofsIFNAR2 (P40-ESEFS) (residues 1-5 of SEQ ID No. 3) is linked to theN-terminal of IFNβ (MSY) via flexible linkers have, in fact, beenprepared. Examples of peptide linkers are as follows:ESEFS(GGGGS)_(n)MSY, where n=5 (SEQ ID NO:3), 4 (SEQ ID NO:4), 3 (SEQ IDNO:5), 2 (SEQ ID NO:6), or 1 (SEQ ID NO:7); ESEFS(hCG-CTP)MSY wherehCG-CTP=SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO:8); ESEFS(EFM)_(n)MSY,where n=5 (SEQ ID NO:9), 4 (SEQ ID NO:10), or 3 (SEQ ID NO:11);ESEFS(EFGAGLVLGGQFM)_(n)MSY where n=1 (SEQ ID NO:12), or 2 (SEQ IDNO:13) and any other suitable linker which spans the distance betweenthe IFNAR2 binding site and IFNβ in the complex model and which do notform an immunogenic epitope between the interferon and receptormoieties. Preferably, these linkers are up to about 30 amino acids inlength.

Covalent Complex. One example of generating chemical crosslinkedmolecules is to site-specifically modify the IFNAR2 by reacting abiodegradable linker, such as polyethylene glycol (PEG), with cysteinespresent or engineered into IFNAR2 using either an amino acidsubstitution, such as. Ser₂₁₀ to Cys or Asn₈₉ to Cys, (site-directedmutagenesis). The following constructs could be engineered:IFNAR(S210C)-PEGn-IFNβ(Cys17) where n=2000, 5000 or 10,000 Daltons orIFNAR(N89C)-PEGn-IFNβ(Cys17).

Formation of covalent disulfide bonds between Cys of the two different(optionally-engineered) moieties is also within the scope of the presentinvention.

Non-Covalent Complex. Human IFNAR2 is complexed with IFNβ underconditions which maximize the generation of the active complex. Invitro, an optimum ratio of 2.5 ng of IFNAR2 to 1 international unit (IU)of IFNβ is required to yield a maximally active complex, as reported inthe examples. The optimum ratio of IFNAR2 to IFNβ which maximizes thegeneration of the active complex for in vivo activity is currently beingdetermined, although it appears that the optimum ratio will be dependenton the concentration of IFNβ. Thus, for example, the optimum ratio ofIFNAR2:IFNβ in the enhancement of anti-tumor activity at a concentrationof 2×10⁴ IU/mouse/day IFNβ was 2.5 ng IFNAR2 per pg IFNβ, while at aconcentration of 5×10⁴ IU/mouse/day IFNβ, 0.3 ng IFNAR2 per pg IFNβ wasoptimum. The same ratio as used to maximize the generation of the activecomplex in vitro resulted in elongated pharmacokinetics of the IFN invivo.

Preferably, IFNAR2 and IFNβ used to generate the complex are recombinantmolecules. In in vitro anti-viral assays, the interferon/receptorcomplex exhibited enhanced activity when compared to the activity ofIFNβ alone. A constant concentration of IFNβ was mixed with varyingconcentrations of recombinant sIFNAR2, and this mixture (IFNAR2/IFNcomplex) was added to WISH cells (human amniotic cells). These WISHcells were then challenged with vesicular stomatitis virus (VSV), andthe anti-viral activity of IFN monitored as the degree of cell survivalfollowing 48-hour incubation. In each of the experiments, the additionof IFNAR2 to a constant amount of IFNβ resulted in a dose-dependentincrease in cell survival upon challenge with VSV at a optimum ratio ofIFNAR2 to IFN. These results prove that a complex of IFNAR2 and IFNβexhibits enhanced activity compared to free IFNβ in an anti-viral assay.The practical implications of this are that the IFNAR2/IFNβ complex hasgreater potency and enhanced activity compared to free IFN for a varietyof therapeutic indications in which IFN by itself is active. Theseindications include those in which free IFNs have shown some therapeuticactivity, such as anti-viral, anti-cancer and immune modulatoryactivity. It is expected that the IFNAR2/IFN complex, by virtue of itsgreater potency, enhanced activity and/or improved pharmacokinetics(i.e. half-life), will be more efficacious in treating viral, oncologicand immune disorders.

When administered in vivo, the interferon receptor complex enhances thebioavailability, pharmacokinetics, and/or pharmacodynamics of the IFN,thus augmenting the anti-viral, anti-cancer and immune modulatingproperties of the IFN.

The enhanced bioavailability of IFN mediated by the complex can begained by either pre-formation of an IFN/IFNAR non-covalent complex,co-administration of free IFN with IFNAR, via administration of the IFNor IFNAR component sequentially, via administration of an IFN/IFNARcovalent complex or via administration of an IFNAR/IFN fusion protein.

In a further embodiment, such enhanced bioavailability may also beaccomplished by administering the IFNAR component alone, without addingany IFN. The IFNAR will form the “complex” in vivo with endogenous IFNand, thus, enhance the bioavailability, pharmacokinetics and/orpharmacodynamics of the endogenous IFN. This is particularly useful forthe treatment of patients having a disease or condition which naturallycauses the induction of native IFN, so that the IFN will already becirculating for its intended natural effect of fighting such disease orcondition. The added IFNAR will potentiate the effects of the nativeIFN.

The preferred molecules for use in the complexes of the presentinvention have the sequence of a native IFN and IFNAR. The nativesequence is that of a naturally occurring human IFN or IFNAR. Suchsequences are known and can be readily found in the literature.Naturally occurring allelic variations are also considered to be nativesequences.

The present invention also concerns analogs of the above IFNAR2/IFNcomplex of the invention, which analogs retain essentially the samebiological activity of the complex having essentially the sequences ofnative IFNAR2 and IFN. Such analogs may be ones in which up to about 30amino acid residues may be deleted, added or substituted by others inthe IFNAR2 and/or IFN moieties of the complex, such that modificationsof this kind do not substantially change the biological activity of thechimeric protein analog with respect to the complex itself. The variousanalogs may differ most from each other and from the basic complexmolecule (that with essentially only naturally occurring IFNAR2 and IFNsequences) at the site of the linker peptide which joins the twomoieties in the complex. As reported above, such a linker is preferablyup to about 30 amino acids in length, and serves to separate the IFNAR2and IFN moieties from each other in the complex. As regards such alinker, care should be taken to choose its sequence (and hence also totest biologically in appropriate standard assays each such analog) suchthat it will, for example, not result in incorrect folding of thecomplex, which may render it inactive or without enhanced activity, orrender the complex analog immunogenic, which will elicit antibodiesagainst it in a patient to be treated therewith with the result thatsuch an analog will be ineffective at least as a medium- or long-termmedicament. As regards the above analogs of the complex of theinvention, these analogs are those in which one or more and up to about30 of the amino acid residues of the basic complex of the invention arereplaced by different amino acid residues, or are deleted, or one ormore amino acid residues, up to about 30, are added to the originalsequence of complex of the invention (that with essentially only thenative IFNAR2/IFN sequences) without changing considerably the activityof the resulting products as compared with basic complex of theinvention. These analogs are prepared by known synthesis and/or bysite-directed mutagenesis techniques or any other known techniquesuitable therefor.

Any such analog preferably has a sequence of amino acids sufficientlyduplicative of that of the basic IFNAR2/IFN complex such as to havesubstantially similar activity thereto. Thus, it can be determinedwhether any given analog has substantially the same activity and/orstability as the basic complex of the invention by means of routineexperimentation, comprising subjecting each such analog to thebiological activity and stability tests set forth in Examples 2-7 below.

Analogs of the complex which can be used in accordance with the presentinvention, or nucleic acid a sequence coding therefor, include a finiteset of substantially corresponding sequences as substitution peptides orpolynucleotides which can be routinely obtained by one of ordinary skillin the art, without undue experimentation, based on the teachings andguidance presented herein. For a detailed description of proteinchemistry and structure, see Schulz. et al, Principles of ProteinStructure, Springer-Verlag, New York (1978); and Creighton, T. E.,Proteins: Structure and Molecular Properties, W. H. Freeman & Co, SanFrancisco (1983), which are hereby incorporated by reference. For apresentation of nucleotide sequence substitutions, such as codonpreferences, see Ausubel et al (1987, 1992), §§A.1. I-A. 1.24, andSambrook et al (1987, 1992), §§6.3 and 6.4, at Appendices C and D.

Preferred changes for analogs in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of those in the complex having essentially thenaturally occurring IFNAR2 and IFN sequences, may include synonymousamino acids within a group which have sufficient similar physicochemicalproperties that substitution between members of the group will preservethe biological function of the molecule (Grantham, 1974). It is clearthat insertions and deletions of amino acids may also be made in theabove-defined sequences without altering their function, particularly ifthe insertions or deletions only involve a few amino acids, e.g., underthirty, and preferably under ten, and do not remove or displace aminoacids which are critical to a functional conformation, e.g., cysteineresidues (Anfinsen, 1973). Analogs produced by such deletions and orinsertions come within the purview of the present invention.

Preferably, the synonymous amino acid groups are those defined in TableI. More preferably, the synonymous amino acid groups are those definedin Table II; and most preferably the synonymous amino acid groups arethose defined in Table III.

TABLE I Preferred Groups of Synonymous Amino Acids Amino Acid SynonymousGroup Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe,Tyr, Met, Val Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His,Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val GlyAla, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met,Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser,Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr,Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu,Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu,Met Trp Trp

TABLE II More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE III Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Glu Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of the complex IFNAR2/IFN for use in thepresent invention include any known method steps, such as presented inU.S. Pat. No. RE 33,653; U.S. Pat. Nos. 4,959,314; 4,588,585 and4,737,462, to Mark et al; U.S Pat. No. 5,116,943 to Koths et al; U.S.Pat. No. 4,965,195 to Namen et al; and U.S. Pat. No. 5,017,691 to Lee,et al, and lysine substituted proteins presented in U.S. Pat. No.4,904,584 (Shaw et al).

In another preferred embodiment of the present invention, any analog ofthe complex for use in the present invention has an amino acid sequenceessentially corresponding to that of the above-noted basic complex ofthe invention. The term “essentially corresponding to” is intended tocomprehend analogs with minor changes to the sequence of the basiccomplex which do not affect the basic characteristics thereof,particularly insofar as its ability to inhibit cancer cell proliferationor promote bone marrow transplantations, for example, is concerned. Thetype of changes which are generally considered to fall within the“essentially corresponding to” language are those which would resultfrom conventional mutagenesis techniques of the DNA encoding the complexof the invention, resulting in a few minor modifications, and screeningfor the desired activity in the manner discussed above.

Preferably, the IFNAR2 portion of the complex will have a core sequencewhich is the same as that of the native sequence or biologically activefragment thereof, or a variant thereof which has an amino acid sequencehaving at least 70% identity to the native amino acid sequence andretains the biological activity thereof. More preferably, such asequence has at least 85% identity, at least 90% identity, or mostpreferably at least 95% identity to the native sequence.

With respect to the IFN portion of the complex, the core sequence whichmay be used is the native sequence, or a biologically active fragmentthereof, or a variant thereof which has an amino acid sequence having atleast 70% identity thereto, more preferably, at least 85% or at least90% identity, and most preferably at least 95% identity. Such analogsmust retain the biological activity of the native IFN sequence orfragment thereof, or have antagonist activity as discussed hereinbelow.

The term “sequence identity” as used herein means that the sequences arecompared as follows. The sequences are aligned using Version 9 of theGenetic Computing Group's GAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of−12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence.

Analogs in accordance with the present invention may also be determinedin accordance with the following procedure. With respect to either theIFNAR portion of the complex or the IFN portion of the complex, the DNAof the native sequence is known to the prior art and is either found inthe literature cited in the background section of the presentspecification or can be readily located by those of ordinary skill inthe art. Polypeptides encoded by any nucleic acid, such as DNA or RNA,which hybridize to the complement of the native DNA or RNA under highlystringent or moderately stringent conditions, as long as thatpolypeptide maintains the biological activity of the native sequence or,in the case of IFN, either maintains the biological activity orpossesses antagonistic activity, are also considered to be within thescope of the present invention.

Stringency conditions are a function of the temperature used in thehybridization experiment, the molarity of the monovalent cations and thepercentage of formamide in the hybridization solution. To determine thedegree of stringency involved with any given set of conditions, onefirst uses the equation of Meinkoth et al. (1984) for determining thestability of hybrids of 100% identity expressed as melting temperatureTm of the DNA-DNA hybrid: Tm=81.5° C.+16.6(_(log)M)+0.41(% GC)−0.61(%form)−500/L where M is the molarity of monovalent cations, %GC is thepercentage of G and C nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. For each 1° C. that the Tm is reduced from thatcalculated for a 100% identity hybrid, the amount of mismatch permittedis increased by about 1%. Thus, if the Tm used for any givenhybridization experiment at the specified salt and formamideconcentrations is 10° C. below the Tm calculated for a 100% hybridaccording to equation of Meinkoth, hybridization will occur even ifthere is up to about 10% mismatch.

As used herein, highly stringent conditions are those which are tolerantof up to about 15% sequence divergence, while moderately stringentconditions are those which are tolerant of up to about 20% sequencedivergence. Without limitation, examples of highly stringent (12-15° C.below the calculated Tm of the hybrid) and moderately (15-20° C. belowthe calculated Tm of the hybrid) conditions use a wash solution of 2×SSC(standard saline citrate) and 0.5% SDS at the appropriate temperaturebelow the calculated Tm of the hybrid. The ultimate stringency of theconditions is primarily due to the washing conditions, particularly ifthe hybridization conditions used are those which allow less stablehybrids to form along with stable hybrids. The wash conditions at higherstringency then remove the less stable hybrids. A common hybridizationcondition that can be used with the highly stringent to moderatelystringent wash conditions described above is hybridization in a solutionof 6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA at a temperature approximately20° to 25° C. below the Tm. If mixed probes are used, it is preferableto use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel,1987, 1998).

“Functional derivatives” as used herein covers derivatives which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C- terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e., they do not destroy the biological activity of thecorresponding protein of the complex as described herein and do notconfer toxic properties on compositions containing it or the complexmade therefrom. Derivatives may have chemical moieties, such ascarbohydrate or phosphate residues, provided such a fraction has thesame biological activity and remains pharmaceutically acceptable.

For example, derivatives may include aliphatic esters of the carboxyl ofthe carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives or freeamino groups of the amino acid residues formed with acyl moieties (e.g.,alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl group (e.g., that of seryl or threonyl residues) formed withacyl moieties. Such derivatives may also include for example,polyethylene glycol side-chains which may mask antigenic sites andextend the residence of the complex or the portions thereof in bodyfluids.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twentycommonly-occurring natural amino acids.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the complex of the invention oranalogs thereof. Salts of a carboxyl group may be formed by means knownin the art and include inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must have substantially similar biologicalactivity to the complex of the invention or its analogs.

The term “biological activity” as used herein is interpreted s follows.Insofar as the IFNAR2 portion of the complex is concerned, the importantbiological activity is its ability to bind to Type I interferon. Thus,analogs or variants, salts and functional derivatives must be thosechosen so as to maintain this interferon-binding ability. This can betested by routine binding assay experiments. In addition, fragments ofthe IFNAR2, or analogs thereof, can also be used as long as they retaintheir interferon-binding activity. Fragments may readily be prepared byremoving amino acids from either end of the interferon-bindingpolypeptide and testing the resultant for interferon-binding properties.Proteases for removing one amino acid at a time from either theN-terminal or the C-terminal of a polypeptide are known, and sodetermining fragments which retain interferon-binding ability involvesonly routine experimentation.

Additionally, the polypeptide which has such interferon-bindingactivity, be it IFNAR2, sINFAR2, an analog or variant, salt, functionalderivative or fragment thereof, can also contain additional amino acidresidues flanking the interferon-binding polypeptide. As long as theresultant molecule retains the interferon-binding ability of the corepolypeptide, one can determine whether any such flanking residues affectthe basic and novel characteristics of the core peptide, i.e., itsinterferon-binding characteristics, by routine experimentation. The term“consisting essentially of”, when referring to a specified sequence,means that additional flanking residues can be present which do notaffect the basic and novel characteristic of the specified sequence.This term does not comprehend substitutions, deletions or additionswithin the specified sequence.

While IFNAR2 or sIFNAR2 have been used throughout this description andin the examples, it should be understood that this is merely thepreferred example and that the IFNAR1 subunit, and particularly itsextracellular domain, may be substituted for IFNAR2 wherever IFNAR2 isreferred to in the present disclosure. IFNAR1 can only be used inconjunction with interferons to which it binds. IFNAR1 is known to bindto IFNα. Any complex using IFNAR1 must be with a species of interferon,and preferably a species of IFNα, to which the IFNAR1 binds.

With respect to the interferon part of the complex of the presentinvention, the biological activity which must be maintained in anyanalog or variant, salt, functional derivative or fragment is theactivity of the interferon relied upon for the intended utility. In mostinstances, this will be the ability to bind to a native cell surfacereceptor and thereby mediate signal production by the receptor. Thus,any such analog, derivative or fragment should maintain such receptoragonist activity to be useful in the present invention for such autility. On the other hand, it is sometimes useful to have a moleculewith antagonist activity on the receptor so as to prevent the biologicalactivity of native interferon. Such an antagonist can also be used forprolonged beneficial effect by means of the complex of the presentinvention. For such utilities in which it is desired to eliminate anundesired effect of interferon, analogs which are still bound by thereceptor and by the IFNAR portion of the complex but which do notmediate a signal and block signal generation by the native interferon onthat receptor, may also be considered to be biologically active for thepurpose of this invention and to be encompassed by the term interferonwhen used with respect to the complexes of the present invention.Straightforward assays can determine whether any such analog maintainssuch receptor agonist activity or has receptor antagonist activity andwould, thus, be useful for one of the utilities of the presentinvention.

The present invention also concerns DNA sequences encoding the abovecomplex of the invention and its analogs, as well as DNA vectorscarrying such DNA sequences for expression in suitable prokaryotic oreukaryotic host cells. The ability to generate large quantities ofheterologous proteins using a recombinant protein expression system hasled to the development of various therapeutic agents, e.g., t-PA and EPO(Edington, 1995). The various expression hosts from which recombinantproteins can be generated range from prokaryotic in origin (e.g.,bacteria) (Olins, 1993), through lower eukaryotes (e.g., yeast) (Ratner,1989) to higher eukaryotic species (e.g., insect and mammalian cells(Reuveny, 1993; Reff, 1993). All of these systems rely upon the sameprinciple—introducing the DNA sequence of the protein of interest intothe chosen cell type (in a transient or stable fashion, as an integratedor episomal element) and using the host transcription, translation andtransportation machinery to over-express the introduced DNA sequence asa heterologous protein (Keown, 1990).

In addition to the expression of native gene sequences, the ability tomanipulate DNA at the nucleotide level has expedited the development ofnovel engineered sequences which, although based on natural proteins,possess novel activities as a result of the alteration in primaryprotein structure (Grazia, 1997).

Moreover, chosen sequences of DNA can be physically linked to generatetranscripts which develop into novel fusion proteins where onceindependent proteins are now expressed as one polypeptide unit (Ibanez,1991). The activity of such fusion proteins can be different, e.g., morepotent, than either of the individual proteins (Curtis, 1991).

Human IFNβ is derived from a production process which uses the mammalianChinese hamster ovary cell (CHO). Type 1 interferons can be expressed ina variety of host cells including bacteria (Utsumi, 1987), insect(Smith, 1983) and human (Christofinis, 1981). Human sIFNAR2 was alsoexpressed using the CHO host cell. Alternatively, soluble receptors,such as sIFNAR2, have also been expressed successfully in bacterialexpression systems (Terlizzese, 1996). The DNA for each gene wasintroduced into the CHO genome using a transfection procedure whichresulted in recombination and integration of the expression vector.Cells which expressed the protein of interest were then isolated,cultured and the protein recovered and purified using standardindustrial practices well known in the art.

The invention also concerns a pharmaceutical composition comprising asactive ingredient an IFNAR2/IFN complex or analogs thereof or mixturesthereof or salts thereof and a pharmaceutical acceptable carrier,diluent or excipient. An embodiment of the pharmaceutical composition ofthe invention includes a pharmaceutical composition for enhanced IFNtype action, in the treatment of viral diseases, in anti-cancer therapy,in immune modulation therapy and other applications of interferons andcytokines related thereto.

The pharmaceutical compositions of the invention are prepared foradministration by mixing the complex or its analogs with physiologicallyacceptable carriers and/or stabilizers and/or excipients, and preparedin dosage form, e.g., by lyophilization in dosage vials. The method ofadministration can be via any of the accepted modes of administrationfor similar agents and will depend on the condition to be treated, e.g.,intravenously, intramuscularly, subcutaneously, by local injection ortopical application, or continuously by infusion, etc. The amount ofactive compound to be administered will depend on the route ofadministration, the disease to be treated and the condition of thepatient. Local injection, for instance, will require a lower amount ofthe protein on a body weight basis than will intravenous infusion.

Free IFNβ has a tendency to oligomerize. To suppress this tendency,present day formulations of IFNβ have an acidic pH, which may cause somelocalized irritation when administered. As IFNAR can serve as astabilizing factor for IFNβ and thereby prevent oligomerization, its usein IFNβ formulations can serve to stabilize the IFNβ and thereby obviatethe necessity of acidic formulations. Accordingly, a non-acidicpharmaceutical composition containing IFNβ and IFNAR, along with otherconventional pharmaceutically acceptable excipients, is also a part ofthe present invention.

The present invention also concerns uses of the complex of the inventionor its analogs or mixtures thereof for anti-viral, anti-cancer andimmune modulation therapy. Specifically, the interferonreceptor-interferon complexes of this invention are useful foranti-viral therapy in such therapeutic indications as chronicgranulomatous disease, condyloma acuminatum, juvenile laryngealpapillomatosis, hepatitis A and chronic infection with hepatitis B and Cviruses.

In particular, the interferon receptor-interferon complexes of thisinvention are useful for anti-cancer therapy in such therapeuticindications as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma,chronic myelogenous leukemia, non-Hodgkins's lymphoma and melanoma.

The interferon receptor-interferon complexes of this invention are alsouseful for immune modulation therapy, such as multiple sclerosis,rheumatoid arthritis, myasthenia gravis, diabetes, AIDS, lupus, etc.

Likewise, the present invention also concerns the complex or analogsthereof or mixtures thereof for use in the preparation of medicamentsfor treating the above-mentioned ailments or for use in the above notedindications.

The present invention will now be described in more detail in thefollowing non-limiting Examples and the accompanying drawings.

EXAMPLES Materials and Methods

ANTI-VIRAL WISH BIOASSAY and COMPLEX GENERATION

A WISH assay was developed based on the protocol of Novick et al (1982).

Materials

WISH cells (ATCC CCL 25)

Vesicular Stomatitis Virus stocks (ATCC V-520-001-522), stored at −70°C.

IFNβ, human recombinant, InterPharm Laboratories LTD 90×10⁶ IU/ml,specific activity: 264.5×10⁶ IU/mg.

Soluble human IFNAR2, 373 μg/ml stock concentration in PBS

WISH Growth medium (MEM high glucose with Earls salts+10% FBS+1.0%L-glutamine+Penicillin/Streptomycin (100 U/ml, 100 μg/ml))

WISH Assay medium (MEM high glucose with Earls salts+5% FBS+1.0%L-glutamine+Penicillin/Streptomycin (100 U/ml, 100 μg/ml), MTT at 5mg/ml in PBS, stored at −70° C.

Methods

Dilute recombinant human IFNβ to 19 IU/ml (4× the predetermined EC₅₀dose) in WISH assay medium.

Starting at 90 μg/ml, make eleven (11) three-fold dilutions of humanrecombinant sIFNAR2 in Eppindorf tubes in WISH assay medium. Twelfthtube contains WISH assay medium only.

Add 25 μl of IFNβ to each well in a flat-bottomed 96-well plate (add 25μl of WISH assay medium alone to one 3×12 well section to control forIFNAR2 effects in the absence of IFNβ).

Add 25 μl of each dilution of the sIFNAR2 (or assay medium only in rowtwelve) in triplicate down the twelve rows of the 96-well plate.

Preincubate IFNβ with sIFNAR2 for 1-4 hours in 37° C. incubator prior tothe addition of WISH cells.

Harvest log growth phase WISH cells with trypsin/EDTA solution, wash inWISH assay medium, and bring to a final concentration of 0.8×10⁶cells/ml.

Add 50 μl of WISH cell suspension (4×10⁴ cells per well) to each well.Final concentration of both IFNβ and IFNAR2 is that which is exposed tothe cells, so that the final concentration of IFNβ is 4.75 IU/ml (1×),and the final concentration of sIFNAR2 in row one is 22.5 μg/ml.

After incubation for 24 hours in a 5% CO₂ humidified incubator, 50 Al ofa 1:10 dilution (in WISH assay medium) of VSV stock (a dosepredetermined to lyse 100% of WISH cells within 48 hours) is added toall wells except for the no virus control wells (these receive an equalvolume of assay medium only).

After an additional 48 hours, 25 μl of MTT solution is added to allwells, after which plates are incubated for an additional 2 hours in theincubator.

Contents of wells are removed by plate inversion, and 200 μl of 100%ethanol is added to wells.

After 1 hour, plates are read at 595 nm using the Soft max Pro softwarepackage and Spectramax spectrophotometer system (Molecular Devices).

All sequencing reactions were performed using the ThermoSequenase™radiolabeled terminator cycle sequencing kit (Amersham Life Science;Cleveland, Ohio). The protocols supplied by the manufacturer were used.All sequencing reactions were analyzed on CastAway™ Precast sequencinggels (Stratagene; LaJolla, Calif.) that contained 6% polyacrylamide and7M urea. Sequencing reactions were loaded in the order A-C-G-T.Autoradiographs of the sequencing gels were read manually. The GeneticsComputer Group Sequence Analysis Software Package and UNIX workstationwere used for DNA sequence analysis.

Example 1

To assess the anti-viral activity of the human IFNAR2/IFNβ complex andhuman IFNAR2Ig-IFNβ complex, a fixed concentration of IFNβ (4.75 IU/ml)was preincubated for 3 hours at 37° C. with human sIFNAR2 (recombinantp40) or human IFNAR2Ig at varying concentrations (0.25-30000 ng/ml) andthen tested in a WISH-VSV cytopathicity assay. In the absence of IFN noanti-viral protection was detected (data not shown).

Anti-viral activity of Type I interferons (used at predetermined EC₅₀concentrations) in the presence of approximately 30 ng/ml sIFNAR2reveals optimal agonist activity with IFNβ, but not alone. Anti-viralactivity was measured by MTT conversion 48 h after VSV challenge.

When IFN was present at an expected ED₅₀ concentration, protection wasobserved (see FIG. 1; absorbance equal to 0.45, no protection absorbanceequals ˜0.0, complete protection absorbance equals ˜1.8)). When IFNAR2or IFNAR2Ig was titrated in at varying concentrations there was ˜4×enhancement in the activity of IFNβ up to 32 ng/ml of IFNAR2 andIFNAR2Ig (see also Example 2 and FIG. 2). Above 32 ng/ml IFNAR orIFNAR2Ig, IFNβ activity decreased as expected, presumably due tocompetition for IFNβ of the sIFNAR with the membrane based IFNAR. Thisexperiment, thus, provides support for the increased potency andenhanced activity of IFNβ in the IFNAR2-IFN complex.

Example 2

The effect of changing the IFNβ concentration on sIFNAR2/IFN complexactivity was examined at different concentrations of IFNβ in addition tothe ED₅₀. As can be seen in FIG. 2, at 4.75 IU/ml IFNβ, IFNAR2preincubation for 1 hour enhanced the activity of IFNβ by ˜2× at aconcentration maximum of ˜32 ng/ml. At each of the higher concentrationsof IFNβ it was not possible to detect enhancement of the IFNβ anti-viralactivity as the activity was already maximal. These results also supportthat an IFNAR2/IFNβ complex has enhanced IFN activity.

Example 3

The kinetics of the IFNAR2/IFNβ complex formation was examined byassessing the anti-viral activity of a sub-effective concentration ofIFNβ (0.95 IU/ml) at various times of preincubation with differentconcentrations of IFNAR2. As seen in FIG. 3, 4 hours of preincubationwere necessary to give enhanced anti-viral activity of IFNβ at thissub-effective dose. Thus, at levels of IFNβ which are inactive bythemselves, addition of IFNAR2 to generate a complex resulted insignificant IFN anti-viral activity. This experiment provides additionalsupport for the IFNAR2/IFNβ complex enhanced IFN activity.

Example 4

It has been established that IFNβ bioactivity rapidly declines followingin vitro reconstitution at 37° C. (physiologic saline buffer pH 7.4).This is, at least in part, due to the formation of IFNβ oligomericstructures which have reduced activity. To test whether IFNAR2 enhancesthe stability of IFNβ at physiologic pH, an experiment was conducted inwhich varying concentrations of IFNβ were incubated alone (in RPMI 1640media, Gibco) or in the same media in the presence of IFNAR2 at aconstant ratio of IFNAR2 to IFNβ (2.5 ng/IU).

IFNβ (500 IU/ml) was preincubated with an equal volume of either sIFNAR2(1.25 μg/ml) or RPMI only, for 3 hours at 37° C. Titration of both IFNβsolutions was performed in WISH assay medium in a 96-well plate prior tothe addition of WISH cells. VSV was added after 24 hours and wasassessed after an additional 48-hour incubation as determined by MTTconversion.

As seen in FIG. 4, IFNβ alone has an ED₅₀ of ˜104 IU/ml, whilepreincubation of IFNR with soluble IFNAR2 results in a significantlyenhanced ED₅₀=˜7 IU/ml. The high ED₅₀ of IFNβ alone may be due to theoligomerization of IFNβ in solution. The above results again show theenhanced stability of IFNβ when complexed with IFNAR2.

Example 5

To assess whether enhanced activity of IFNβ in the presence of IFNAR2 isdue to specific protection by sIFNAR, the activity of IFNβwas evaluatedfollowing complexation with IFNAR2 or other unrelated proteins at thesame concentration (human IgG, bovine serum albumin (BSA)).

As shown in FIG. 5, IFNβ (500 IU/ml) was preincubated with an equalvolume of either the indicated proteins (1.25 μg/ml) or RPMI only, for 4hours at 37° C. Titration of these IFNβ solutions in WISH assay mediumwas performed in a 96-well plate prior to the addition of WISH cells.Freshly-prepared IFNβ was also included to determine the effect of thepreincubation on IFNβ activity. VSV was added after 24 hours, and CPEwas assessed after an additional 48 hour incubation as determined by MTTconversion.

Preincubation of IFNβ with non-specific proteins (i.e., BSA or humanIgG) at 2.5 ng/IU IFNβ did not protect the activity of IFNβ afterreconstitution. The activity of the IFNAR2/IFNβ complex in this assaywas similar to freshly added IFNβ, which supports the finding thatsIFNAR2 stabilizes IFNβ activity.

Example 6

IFNAR2/IFNβ complex showed a greatly prolonged pharmacokinetic profileof IFNβ in the mouse based on ELISA and bioassay analysis (FIGS. 6 and7).

B6D2F1 strain mice received a single intravenous bolus injection ofeither human IFNβ (2.5×10⁶ IU/kg) or the same concentration of IFNβpreincubated for 1 hour at 4° C. with human IFNAR2 (2.5 ng/IU of IFN).Sera were collected 0.05 to 48 hours post-administration from theretro-orbital plexus, and IFNβ concentration and IFNβ anti-viralactivity was assessed by ELISA (FIG. 6) or WISH bioassay (FIG. 7),respectively. Serum concentrations falling below the level of assaysensitivity (7.55 IU/ml) in the ELISA assay were not plotted.

Not only did the complex show an extended pharmacokinetic profile, butby WISH anti-viral assay the level of biologically active IFNβ in themouse was greatly enhanced over time, thus showing the enhancedstability and elongated plasma half-life of the IFNAR2/IFNβ complex ofthe invention with respect to IFNβ alone.

Example 7

IFNAR2/IFNα2a complex showed a greatly prolonged pharmacokinetic profileof IFNα2a in the mouse based on ELISA and bioassay tests (FIGS. 8 and9).

B6D2F1 strain mice received a single intravenous bolus injection ofeither human IFNα (1.25×10⁵ IU/kg) or the same concentration of IFNαpreincubated for 1 hour at 4° C. with human IFNAR2 (14.9 ng/IU of IFN).Sera were collected at indicated times from the retro-orbital plexus,and IFNα2a concentration and IFNα anti-viral activity was assessed byELISA (FIG. 8) or WISH bioassay (FIG. 9), respectively.

IFNα was assessed by ELISA specific for human IFNα. Serum concentrationsfalling below the level of assay sensitivity (30 IU/ml) in the ELISAwere not plotted.

Not only did the complex show an extended pharmacokinetic profile forIFNα, but by WISH anti-viral assay the level of biologically active IFNαin the mouse was greatly enhanced over time, thus showing the enhancedstability and elongated plasma half-life of the IFNAR2/IFN complex ofthe invention with respect to IFNα alone.

Example 8

Engineering of IFNAR2/IFN Fusion Proteins. Constructs were engineered sothat the C-terminal of the IFNAR2 extracellular domain is fused to theN-terminus of the mature IFN using the following peptide linkers, with Gand S representing the amino acids glycine and serine, respectively:

IFNAR2extracellular (linker) mature IFNβ

. . . ESEFS(GGGGS)_(n)MSY . . . , where n=0 (SEQ ID NO:15), 1 (SEQ IDNO:7), 2 (SEQ ID NO:6), 3 (SEQ ID NO:5), 4 (SEQ ID NO:4), and 5 (SEQ IDNO:3)

The full amino acid sequence of the n=2 IFNAR2/IFNβ fusion is shown inFIG. 10 (SEQ ID NO:14).

The expression vector pSVEIF, which contained the gene expressing humanrecombinant IFN, was used as the template for PCR. Synthetic primerswere designed so that only the mature protein coding region of human IFN(MSY . . . ) could be amplified from the template. The 5′ primerconsisted of sequences for a digested-SmaI site, the last seven aminoacids of IFNAR2 (GQESEFS) (residues 344-349 of SEQ ID NO:14), and the(GGGGS)₁ (SEQ ID NO:1) linker. BamHI and XhoI sites were also introducedin the 5′ PCR primer to facilitate cloning of the other cassettes. The3′ primer contained an AvrII site immediately following the TGA stopcodon of hIFNβ. The PCR contained approximately 1 g of template DNA, 1 gof each PCR primer, 0.2 mM each of dATP, dCTP, dGTP, and dTTP,1×ThermoPol Reaction Buffer (10 mM KCl, 20 mM Tris-HCl, (pH 8.8 at 25°C.), 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100: New EnglandBiolabs; Beverly, Mass.), and 3 mM MgSO₄ (5 mM MgSO₄ finalconcentration) in a reaction volume of 100 μl. After an VENTR® initialincubation at 99.9° C. for 30 seconds, 2 units of VENTR® DNA polymerasewas added to the reaction. PCR consisted of 20 cycles of the following:(a) 99.9° C. for 30 seconds, (b) 65° C.-55° C. for 30 seconds,decreasing 0.5° C. with each cycle, and (c) 75° C. for 40 seconds. Anadditional 15 cycles were done with the above profile but with theannealing temperature held at 55° C. The PCR reactions were purifiedusing the Wizard™ PCR Preps DNA Purification System (Promega; Madison,Wis.). After digestion of the PCR products with AvrII, the reactionswere purified on low melting point agarose gels. The gel-purifiedfragment containing the hIFNβ mature protein sequence with 1 GS linkerwas ligated into the (SmaI+AvrII)-digested expression vector pCMV-p40,which contained the gene coding for the soluble form of humanrecombinant IFNAR2. The ligation reactions were used to transformcompetent E. coli XL-1 Blue cells using standard methods (Sambrook etal, 1989). Correct assembly of the construction, called pCMV-IFNAR2-IFNαGS, was confirmed by restriction endonuclease digestion and sequencingof the PCR-generated region of the “Interfusion”. Subsequent constructswere engineered by using oligonucleotide cassettes, each containing aBamHI overhang, the appropriate (GGGGS)_(n) linker (SEQ ID NO: 1 whenn=1) and a XhoI overhang. The 0 GS cassettes contained SmaI and XhoIoverhangs. After confirmation of the pCMV-IFNAR2/IFNAβ 1 GS vector, itwas digested with BamHI and XhoI, and the appropriate cassettes wereligated into this vector. For the 0 GS construct, the vector wasdigested with SmaI and XhoI for ligation of the cassette.

A total of six vectors were created, pCMV-IFNAR2/IFNβ n (GS), where nrepresents 0, 1, 2 ,3, 4, or 5 GGGGS (SEQ ID NO:1) linker units. Theserestriction digestion results are shown in FIG. 11. Sequencing primerswere designed so that the cassette for each construct was sequenced infull. Large scale plasmid DNA cultures were prepared for each of theconfirmed constructs using a commercially available kit and theprotocols described by the manufacturer (Qiagen; Chatsworth, Calif.).

FIG. 12 is a representative plasmid map of the pCMV-IFNAR2/IFNAβ“Interfusion” expression vectors. Transcription of the IFNAR2/IFNβfusion protein is directed by the human immediate early CMV promoter.The human growth hormone (hGH) polyadenylation signal sequence providedby the vector was used for 3′ processing of the IFNAR2/IFNβ transcripts.

List of Vectors GGGGS (SEQ ID NO: 1) No. Name Exp. Vector Linker 1pCMV-IFNAR2-IFNβ, 0GS pCMV.PA4 N/A 2 pCMV-IFNAR2-IFNβ, 1GS pCMV.PA4 One3 pCMV-IFNAR2-IFNβ, 2GS pCMV.PA4 Two 4 pCMV-IFNAR2-IFNβ, 3GS pCMV.PA4Three 5 pCMV-IFNAR2-IFNβ, 4GS pCMV.PA5 Four 6 pCMV-IFNAR2-IFNβ, 5GSpCMV.PA4 Five

Sequence data was obtained for the PCR-generated region of IFNAR2/IFNβ1GS fusion; this data showed that the sequence was as predicted.Sequence data was obtained for the peptide linker region of the otherconstructions; the sequences were also as predicted.

A Northern blot analysis was performed on cells transfected with eachconstruct. A band of approximately 1.4 kb in size was present in alllanes containing the IFNAR2/IFNβ n GS samples for both the IFNAR2 probeand for the IFNβ probe. With the IFNβ probe, an additional band ofapproximately 0.9 kb is observed. A band of this size would correspondto an alternatively spliced transcript that would contain the last sixamino acids of IFNAR2, the (n) GS peptide linker, and the hIFN matureprotein coding sequence. This was confirmed by sequencing cDNA preparedfrom the total RNA isolated from the transiently transfected CHO cellsof Example 9.

Example 9

Transient Transfection. CHO-DUKX cells are a clonal mutant of Chinesehamster ovary cells lacking dihydrofolate reductase activity (Urlaub etal (1980); Graff et al (1982)). The cells were maintained in AlphaMinimum Essential Medium (αMEM) plus ribonucleosides anddexoyribonucleosides, supplemented with 10fetal bovine serum (FBS) and1% L-glutamine (complete medium). A transient transfection was doneusing the Lipofectamine PLUS™ Reagent (GibcoBRL Life Technologies;Gaithersburg, Md.) and the protocol provided by the manufacturer.Approximately 24 hours prior to transfection, cells were plated in 100mm diameter dishes at a density of 2×10⁶ cells/dish. For thetransfection, 4 μg of the supercoiled vector plasmid DNA(pCMV-IFNAR2-IFN n GS, where n=0, 1, 2, 3, 4, or 5) was used. Theserum-free medium provided by the manufacturer was used for dilution ofthe DNA and PLUS reagent. Cell supernatants were collected afterincubation at 37° C. for 48 hours in complete medium for ELISAs (IFNAR2,IFNβ) and Western gels.

RNA Extraction and Northern Analysis. Total cellular RNA (Chomczynski etal, 1987) was extracted from the transiently transfected CHO-DUKX cells.Ten g of total RNA per lane was size-fractionated in agarose gels whichcontained formaldehyde as a denaturant. Samples were loaded in duplicatesets. The RNA was transferred to GeneScreen Plus nylon membranes(DuPont/NEN Medical Products; Boston, Mass.) by capillary blot in 10×SSC(1.5M sodium chloride, 0.15M sodium citrate). The immobilized RNA washybridized to ³²P-labeled hIFNAR2 and hIFNβ PCR fragments in a solutionmodified from that of Church and Gilbert (1984). The buffer contained0.25M sodium phosphate, pH 7.2, 0.25M sodium chloride, 7% sodium dodecylsulfate (SDS), 1 mM ethlylenediaminetetraacetic acid and 100 μg/ml E.coli tRNA. The ³²P-labeled probes were generated using a commerciallyavailable kit (“High Prime” Boehringer Mannheim; Indianapolis, Ind.) andthe procedures described therein. Non-incorporated radioactivity wasremoved by chromatography on Sephadex G-50 columns. After hybridization,the blots were washed; the most stringent condition used was 0.2×SSC,0.1% SDS at 65° C. The blots were subjected to autoradiography.

Expression of the Interfusion Proteins. Supernatants from each of theInterfusion constructs transiently transfected into CHO cells wereanalyzed for IFNAR2 and IFNβ expression levels using an IFNAR2 specificELISA and an IFNβ ELISA (Toray), respectively. The results of thisanalysis are shown below in Table IV.

TABLE IV Interfusion Constructs Transiently Transfected into CHO CellsSample [hIFNβ]*, [hIFNAR2]^(†), [hIFNβ]^(‡), [hIFNβ]^(#), [hIFNAR2]^(§),Identification (U/ml) (ng/ml) (μg/ml) (pmols) (pmols) IFN/IFNAR 0GS29,175 1094  0.146 6.571 31.802 0.207 1GS 24,906 994 0.125 5.609 28.8950.194 2GS 13,597 600 0.068 3.063 17.442 0.175 3GS 14,998 718 0.075 3.37820.857 0.162 4GS  9,998 535 0.050 2.251 15.538 0.145 5GS  9,597 5400.048 2.161 15.698 0.138 IFNAR2 Not Found 1176  34.200 Culture MediumNot Found  0 *Determined using the TORAY kit ^(†)Determined using thehIFNAR2 ELISA ^(‡)Based on a specific activity of 2.0 × 10⁵ U/mg (2 ×10⁸/mg) ^(#)Based on a mass of 22,200 daltons (average mass by MALDITOFanalysis of hIFNβ) ^(§)Based on a mass of 34,400 daltons (average massby MALDITOF analysis)

As can be seen, with increasing linker length a decrease in detectableIFNAR2 and IFNβ was observed. At this time it is not possible toestablish whether this is due to a decrease in the amount of Interfusionprotein expressed as the linker length increases or whether, as thelinker length increases, the IFNβ can bind in the IFNAR2 binding siteand, therefore, is not completely detectable by the ELISA assays. It isknown that the IFNβ assay only detects non-complexed IFNβ.

Western Blot Analysis of IFNAR2/IFNβ Fusion Protein. In order toestablish that the Interfusion proteins were being expressed at theappropriate molecular weight and that no free IFNβ was being expressed,supernatants from transfected cells were analyzed by Western blottingusing an anti-IFNβ antibody to detect the Interfusion. The results ofthis analysis are shown in FIG. 13.

15 μl of culture supernatants from CHO cells transiently transfectedwith IFNAR2/IFNβ construct (lanes 1-6) or IFNAR2 construct (lane 7),culture medium (lane 8) or IFNβ (lane 9) were subjected to SDS-PAGEunder non-reducing condition followed by electrotransfer to PVDFmembrane. The membrane was probed with rabbit anti-IFNβ antibody anddeveloped with alkaline phosphatase conjugated goat anti-rabbit using aWestern-Star luminescence detection kit.

No free IFNβ was detected in the supernatants of any of the Interfusionconstructs. Likewise each of the constructs expressed a protein that byWestern blot was the appropriate MW for each Interfusion construct.

Example 10

Anti-viral Activity of Interfusion Molecules. Each Interfusioncontaining culture supernatant was tested for anti-viral activity in acytopathicity assay in which WISH cells (human amniotic cells) wereexposed to VSV following the addition of either IFNβ (control) or theInterfusions. The results are shown in FIG. 14.

CHO cell supernatants containing expressed recombinant proteins (asdetected by ELISA and Western Blot), or CHO culture medium alone, wereadded in duplicate to the top row of 96-well flat-bottom tissue cultureplates in a volume of 75 μl/well. 50 μl of WISH cell assay medium wasadded to the remaining wells of the plate. Three-fold serial dilutionsof each sample were performed by removing 25 μl from the wellscontaining the supernatants (top row) and adding this to the next rowcontaining the 50 μl of the WISH assay medium. In the positive controlwells, the supernatant in the top row was replaced with WISH assaymedium containing 1000 IU/ml human IFNβ, which was likewise seriallydiluted three-fold down the length of the plate. Each well then received50 μl of a WISH cell suspension (0.6×10⁶ cells/ml in WISH assay medium)so that the final concentration in the top row containing REBIF® is 500IU/ml, and the starting dilution for the CHO cell supernatants is 1:2.Following 24 hours of incubation in 5% CO₂ at 37° C., each well (exceptthose designated as the uninfected control wells) received 50 μl of WISHassay medium containing vesicular stomatitis virus (1:10 of the stock).Viability of the WISH cells was determined, following an additional 48hours of culture, by MTT conversion.

Anti-viral activity mediated by the Interfusion molecules was determinedby normalizing the dilutional factor of the supernatants necessary toachieve the EC₅₀ to the EC₅₀ determined for the purified human IFNαstandard. As the linker length increases, so too does the anti-viralactivity of the Interfusion constructs.

Example 11

This example is a pharmacokinetic study of human IFNβ/sIFNAR2 complex inthe mouse upon intravenous administration. Comparisons are performedbetween preformed and separately injected complex components. Thirty-sixD2F1 strain mice (6-8 wks) (approximately 20 g each) are separated intofour groups as follows:

Group 1 contains nine mice to be injected intravenously with a singlebolus of 200 μl of a solution of 50,000 IU/ml human IFNβ (final dose of10,000 IU/mouse).

Group 2 (nine mice) received 200 μl of a solution of 50,00 IU/ml humanIFNβ and 125 mg/ml sIFNAR2 (2.5 ng/IU ratio).

Group 3 (nine mice) received (1) 200 μl of a solution of 125 mg/ml,followed by (2) 200 ml of a solution of 50,000 IU/ml human IFNβ (2.5ng/IU ratio).

Group 4 (9 mice) received (1) 200 μl of a solution of 625 mg/ml,followed by (2) 200 ml of a solution of 50,000 IU/ml human IFNβ (10ng/IU ratio).

Blood samples (approximately 200 μl/sample) are collected at thespecified times by disruption of the retro-orbital venous plexus with acapillary tube. Three mice of each group have blood samples taken at0.05, 2 and 12 hours post administration. Three mice of each group haveblood samples taken at 0.54 and 24 hours post administration and threeof each group have blood samples taken at 1, 8 and 48 hours postadministration. Blood samples were allowed to clot at one hour at roomtemperature rimmed and microcentrifuged. Sera removed therefrom werestored at −70° C. until all samples were collected. Sera are assayed forthe presence of human IFNβ by means of IFNβ specific ELISA using theToray human IFNβ ELISA kit (TFB, Inc.) and were assayed for bioactivityusing the WISH antiviral assay.

The results of the IFNβ specific ELISA assay are shown in FIG. 15A andthe results of the WISH antiviral assay are shown in FIG. 15B.

It can be seen that the serum half life of IFNβ injected as IFNAR2complex is similar to that of IFNβ injected following separate IFNARinjection. These results are consistent with an in vivo formation of anIFNβ/IFNAR2 complex with enhanced half life.

Example 12

C57BL/6 mice were treated with a complex of “Universal” IFN (humanIFNαA/D) and sIFNAR2. The protective effect in terms of cytotoxicity wasmeasured as compared with administration of various doses of UniversalIFN alone or a control. The first group received 5×10³ IU Universal IFN,complexed with 5/ng IU sIFNAR2. The second group received 5×10³ IUUniversal IFN. The third group received 5×10⁴ IU Universal IFN and thefourth group received PBS/2% NMS. For each of these mice, NK activitywas measured as cytotoxicity of splenic cells against the NK targetcells YAC-1. The results are shown in FIG. 16. The NK activity wassignificantly greater in mice treated with the Universal IFN/sIFNAR2complex as compared with mice treated with Universal IFN only.

Example 13

As indicated above, pharmacokinetic studies have demonstrated a dramaticenhancement in the serum half life of Type I IFNs when administered as acomplex with sIFNAR2, the soluble form of the IFN receptor subunits. Invitro results suggest that physical association with IFNAR2 leads to thestabilization of normally labile IFN. In order to determine whether theenhanced PK profile and stabilizing effect of IFNAR2 cause anenhancement and prolongation of IFN mediated efficacy in vivo, a modelwas developed in which severe combined immunodeficient (scid/scid) miceare challenged with a lethal does of the IFN-sensitive Daudi human Bcell lymphoma cell line (Ghetie, 1991; Ghetie, 1990). These micedeveloped paralysis between days 14-20 post tumor cell injection inassociation with histological evidence of diffused lymphoma. Notably,survival of these mice can be prolonged in dose dependent fashion bydaily subcutaneous administration of human IFNβ. This model is beingused to evaluate IFNAR2 as a potentiator of the biological activityassociated with type 1 IFNs in vivo.

In order to establish the relationship between the mean time toparalysis and the dose of IFNβ in the Daudi scid model, five groups of 5BALB/cByJSmn-scid/scid strain mice, 4-5 wks of age, female, received asubcutaneous administration of 200 μl per mouse per day of human IFNβevery day from day 0 to day 30. The standard Daudi cell dose, expandedfrom frozen stock, was 5×10⁶ cells per mouse by subcutaneous injectionin the scruff of the neck on day 0 in PBS. The groups of mice receivedthe following amounts of IFN.

Group 1: 135×10⁴ IU/mouse (675×10⁴ IU/ml).

Group 2: 45×10⁴ IU/mouse (225×10⁴ IU/ml).

Group 3: 15×10⁴ IU/mouse (75×10⁴ IU/ml).

Group 4: 5×10⁴ IU/mouse (25×10⁴ IU/ml).

Group 5: PBS with 2% NMS

The time to paralysis as individual and mean values is shown in Table V.

TABLE V Days to Paralysis Mean (± SD) Group 1 26, 31, 35, 37, 40 33.8(5.4) Group 2 20, 23, 23, 23, 26 23.0 (2.1) Group 3 20, 20, 22, 22, 2321.4 (1.3) Group 4 17, 18, 18, 18, 18 17.8 (0.5) Group 5 14, 14, 15, 15,15 14.6 (0.6)

It can thus be seen that the mean time to paralysis in the Daudi/scidxenograft model is prolonged by daily subcutaneous administration ofhuman IFNβ in a dose dependent manner.

Example 14

In order to determine whether the antitumor effect of IFNβ can beenhanced by complexing with IFNAR2 at 2.5 ng/IU, seven groups of fivemice were treated by the same protocol discussed in Example 13, exceptthat the test materials administered to each of the groups was asfollows:

IFNβ only/mouse IFNβ plus sIFNAR2/mouse Group 1 2 × 10² IU Group 4 2 ×10² IU plus 0.5 μg Group 2 2 × 10³ IU Group 5 2 × 10³ IU plus 5.0 μgGroup 3 2 × 10⁴ IU Group 6 2 × 10⁴ IU plus 50.0 μg Group 7 receivedDaudi cells, treament is dilluent only

The time to paralysis as individual and mean values is shown in thefollowing table:

TABLE VI Days to Paralysis Mean (± SD) Group 1 16, 16, 17, 18, 19 17.2(1.3) Group 2 17, 18, 18, 19, 19 18.2 (0.8) Group 3 17, 17, 17, 18, 1817.6 (0.9) Group 4 17, 17, 18, 18, 19 17.8 (0.8) Group 5 17, 18, 19, 20,20 18.8 (1.3) Group 6 21, 22, 22, 23, 26  22.8 (1.9)* Group 7 16, 16,17, 17, 17 16.6 (0.6) *Significantly different (p value ≦ 0.05) thansame concentration of IFNβ in non-complexed form in pairwise groupcomparison as determined by one-way ANOVA.

It can be seen that the anti-tumor activity of a low dose of IFNβ 2×10⁴IU/mouse/day and the Daudi/scid xenograft model is significantlyenhanced by complexing with IFNAR2.

In an additional experiment (not shown) the effect of injectionfrequency on mean paralysis time was studied. It was determined thatsignificantly enhanced antitumor activity in the Daudi/scid xenograftmodel can be obtained by treatment with IFNβ/IFNAR G2 complex wheninjected as infrequently as once per week, as compared with free IFNβinjected as often as once per day. Furthermore, in an additionalexperiment (not shown) the optimum ratio of IFNAR to IFNβ was tested. Itwas found that the optimum ratio of IFNAR2:IFNβ in the enhancement ofantitumor activity in a single concentration of IFNβ(2×10⁴/IU/mouse/day) was 2.5 ng IFNAR2 per pg IFNβ.

In a second experiment, it was found that the optimum ratio ifIFNAR2:IFNβ in the enhancement of antitumor activity at a concentrationof IFNβ 5×10⁴ IU/mouse/day was 0.3 ng IFNAR2 per pg IFNβ. These twoexperiments indicate that the optimum ratio depends on the concentrationof IFNα and seems to indicate that the higher the concentration of IFNβ,the lower the ratio needs to be.

In another experiment using the same model, it was established thatadministration of IFNAR2 alone does not enhance survival of the mice inthe study.

Example 15

This example is a pharmacokinetic study to determine the serum half lifeof the Interfusion 5GS molecule in mouse serum following a single bolusintravenous injection. Twenty-one female B6D2F1 strain mice (6-8 wks)(approximately 20 g each) were separated into three groups as follows:

Group 1: contains nine mice injected intravenously with a single bolusof 200 μl of a solution of 100,000 IU/ml Interfusion 5GS (final dose of20,000 IU/mouse or 5×10⁶ IU/kg).

Group 2: (nine mice) received 200 μl of a solution of 100,000 IU/mlhuman IFNβ.

Group 3: contains three uninjected mice which serve as a negativecontrol.

Assuming a blood volume of approximately 2 ml/mouse, the theoreticalC_(max) and T_(max) is 10,000 IU/ml for Groups 1 and 2. Three mice ofeach of Groups 1 and 2 had blood sampled at 0.05, 2 and 12 hours postadministration. Three mice of each of Groups 1 and 2 had blood sampledat 0.5, 4 and 24 hours post administration, and three mice of each ofGroups 1 and 2 had blood sampled at 1, 8 and 48 hours postadministration. Sera were assayed for the presence of bioactive humanIFNβ using the WISH assay.

The results are shown in FIG. 17. While IFNβ is cleared almostimmediately, the Interfusion molecule remains in the serum for longafter injection. This shows that the fusion protein has the desiredstabilizing effect.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materialsand steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention. Thusthe expressions “means to . . . ” and “means for . . . ”, or any methodstep language, as may be found in the specification above and/or in theclaims below, followed by a functional statement, are intended to defineand cover whatever structural, physical, chemical or electrical elementor structure, or whatever method step, which may now or in the futureexist which carries out the recited function, whether or not preciselyequivalent to the embodiment or embodiments disclosed in thespecification above, i.e., other means or steps for carrying out thesame functions can be used; and it is intended that such expressions begiven their broadest interpretation.

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15 1 5 PRT Artificial Sequence Description of Artificial Sequence Linker1 Gly Gly Gly Gly Ser 1 5 2 4 PRT Artificial Sequence Description ofArtificial Sequence Factor Xa cleavage recognition signal 2 Ile Glu GluArg 1 3 33 PRT Artificial Sequence Description of Artificial Sequence Cterminal human sIFNAR2 linked by linker to N terminal human IFNbeta 3Glu Ser Glu Phe Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 1015 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser 20 2530 Tyr 4 28 PRT Artificial Sequence Description of Artificial Sequence Cterminal human sIFNAR2 linked by linker to N terminal human IFNbeta 4Glu Ser Glu Phe Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 1015 Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Tyr 20 25 5 23 PRTArtificial Sequence Description of Artificial Sequence C terminal humansIFNAR2 linked by linker to N terminal human IFNbeta 5 Glu Ser Glu PheSer Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly GlySer Met Ser Tyr 20 6 18 PRT Artificial Sequence Description ofArtificial Sequence C terminal human sIFNAR2 linked by linker to Nterminal human IFNbeta 6 Glu Ser Glu Phe Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser Met 1 5 10 15 Ser Tyr 7 13 PRT Artificial SequenceDescription of Artificial Sequence C terminal human sIFNAR2 linked bylinker to N terminal human IFNbeta 7 Glu Ser Glu Phe Ser Gly Gly Gly GlySer Met Ser Tyr 1 5 10 8 36 PRT Artificial Sequence Description ofArtificial Sequence C terminal human sIFNAR2 linked by hCG-CTP linker toN terminal human IFNbeta 8 Glu Ser Glu Phe Ser Ser Ser Ser Ser Lys AlaPro Pro Pro Ser Leu 1 5 10 15 Pro Ser Pro Ser Arg Leu Pro Gly Pro SerAsp Thr Pro Ile Leu Pro 20 25 30 Gln Met Ser Tyr 35 9 23 PRT ArtificialSequence Description of Artificial Sequence C terminal human sIFNAR2linked by linker to N terminal human IFNbeta 9 Glu Ser Glu Phe Ser GluPhe Met Glu Phe Met Glu Phe Met Glu Phe 1 5 10 15 Met Glu Phe Met MetSer Tyr 20 10 20 PRT Artificial Sequence Description of ArtificialSequence C terminal human sIFNAR2 linked by linker to N terminal humanIFNbeta 10 Glu Ser Glu Phe Ser Glu Phe Met Glu Phe Met Glu Phe Met GluPhe 1 5 10 15 Met Met Ser Tyr 20 11 17 PRT Artificial SequenceDescription of Artificial Sequence C terminal human sIFNAR2 linked bylinker to N terminal human IFNbeta 11 Glu Ser Glu Phe Ser Glu Phe MetGlu Phe Met Glu Phe Met Met Ser 1 5 10 15 Tyr 12 21 PRT ArtificialSequence Description of Artificial Sequence C terminal human sIFNAR2linked by linker to N terminal human IFNbeta 12 Glu Ser Glu Phe Ser GluPhe Gly Ala Gly Leu Val Leu Gly Gly Gln 1 5 10 15 Phe Met Met Ser Tyr 2013 34 PRT Artificial Sequence Description of Artificial Sequence Cterminal human sIFNAR2 linked by linker to N terminal human IFNbeta 13Glu Ser Glu Phe Ser Glu Phe Gly Ala Gly Leu Val Leu Gly Gly Gln 1 5 1015 Phe Met Glu Phe Gly Ala Gly Leu Val Leu Gly Gly Gln Phe Met Met 20 2530 Ser Tyr 14 415 PRT Artificial Sequence Description of ArtificialSequence Human sIFNAR2 linked by linker to human IFNbeta 14 Met Leu LeuSer Gln Asn Ala Phe Ile Val Arg Ser Leu Asn Leu Val 1 5 10 15 Leu MetVal Tyr Ile Ser Leu Val Phe Gly Ile Ser Tyr Asp Ser Pro 20 25 30 Asp TyrThr Asp Glu Ser Cys Thr Phe Lys Ile Ser Leu Arg Asn Phe 35 40 45 Arg SerIle Leu Ser Trp Glu Leu Lys Asn His Ser Ile Val Pro Thr 50 55 60 His TyrThr Leu Leu Tyr Thr Ile Met Ser Lys Pro Glu Asp Leu Lys 65 70 75 80 ValVal Lys Asn Cys Ala Asn Thr Thr Arg Ser Phe Cys Asp Leu Thr 85 90 95 AspGlu Trp Arg Ser Thr His Glu Ala Tyr Val Thr Val Leu Glu Gly 100 105 110Phe Ser Gly Asn Thr Thr Leu Phe Ser Cys Ser His Asn Phe Trp Leu 115 120125 Ala Ile Asp Met Ser Phe Glu Pro Pro Glu Phe Glu Ile Val Gly Phe 130135 140 Thr Asn His Ile Asn Val Met Val Lys Phe Pro Ser Ile Val Glu Glu145 150 155 160 Glu Leu Gln Phe Asp Leu Ser Leu Val Ile Glu Glu Gln SerGlu Gly 165 170 175 Ile Val Lys Lys His Lys Pro Glu Ile Lys Gly Asn MetSer Gly Asn 180 185 190 Phe Thr Tyr Ile Ile Asp Lys Leu Ile Pro Asn ThrAsn Tyr Cys Val 195 200 205 Ser Val Tyr Leu Glu His Ser Asp Glu Gln AlaVal Ile Lys Ser Pro 210 215 220 Leu Lys Cys Thr Leu Leu Pro Pro Gly GlnGlu Ser Glu Phe Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly GlySer Met Ser Tyr Asn Leu Leu Gly 245 250 255 Phe Leu Gln Arg Ser Ser AsnPhe Gln Cys Gln Lys Leu Leu Trp Gln 260 265 270 Leu Asn Gly Arg Leu GluTyr Cys Leu Lys Asp Arg Met Asn Phe Asp 275 280 285 Ile Pro Glu Glu IleLys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala 290 295 300 Ala Leu Thr IleTyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg 305 310 315 320 Gln AspSer Ser Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu 325 330 335 LeuAla Asn Val Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu 340 345 350Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser 355 360365 Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala 370375 380 Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu385 390 395 400 Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr Leu ArgAsn 405 410 415 15 8 PRT Artificial Sequence Description of ArtificialSequence C terminal human sIFNAR2 directly connected to N terminal humanIFNbeta 15 Glu Ser Glu Phe Ser Met Ser Tyr 1 5

What is claimed is:
 1. A method for prolonging the in vivo effect ofType I interferon (IFN), comprising: administering to a patient in needof Type I IFN therapy a complex of Type I IFN and a subunit of the humaninterferon α/β receptor (IFNAR) which is capable of binding to the TypeI IFN of the complex, in an amount effective to provide such IFNtherapy, wherein said Type I IFN has a sequence consisting essentiallyof the sequence of a) a native Type I IFN; b) a fragment of a) which hasType I IFN receptor agonist or antagonist activity; c) a variant of a)or b) which has at least 70% sequence identity with a) or b) and whichhas Type I IFN receptor agonist or antagonist activity; or d) a variantof a) or b) which is encoded by a DNA sequence which hybridizes to thecomplement of the native DNA sequence encoding a) or b) under moderatelystringent conditions and which has Type I IFN receptor agonist orantagonist activity; or a salt or functional derivative of a), b), c),or d) which has Type I IFN receptor agonist or antagonist activity; andwherein said IFNAR has a sequence consisting essentially of the sequenceof e) a native human IFNAR polypeptide chain; f) a fragment of e) whichhas IFNAR receptor agonist or antagonist activity; g) a variant of e) orf) which has at least 70% sequence identity with e) or f) and which hasIFNAR receptor agonist or antagonist activity; h) a variant of e) or f)which is encoded by a DNA sequence which hybridizes to the complement ofthe native DNA sequence encoding e) or f) under moderately stringentconditions and which has IFNAR biological activity; or a salt orfunctional derivative of e), f), g), or h) which has IFNAR biologicalactivity, with the proviso that when said Type I IFN and said IFNAR areadministered separately and said complex is formed in vivo, the amountof IFNAR administered is an amount effective to prolong the in vivoeffect of the Type I IFN.
 2. A method in accordance with claim 1,wherein said complex comprises a non-covalent complex of said Type I IFNand said IFNAR.
 3. A method in accordance with claim 2, wherein saidType I IFN and said IFNAR are administered separately and said complexis formed in vivo.
 4. A method in accordance with claim 1, wherein saidcomplex comprises a complex in which said Type I IFN is bound to saidIFNAR by a covalent bond.
 5. A method in accordance with claim 1,wherein said complex comprises a fusion protein in which said Type I IFNis bound to said IFNAR by a peptide bond.
 6. A method in accordance withclaim 5, wherein said Type 1 IFN is linked to said IFNAR by means of apeptide linker.
 7. A method in accordance with claim 1, wherein saidType I IFN is an IFNα, an IFNβ, or an IFNω.
 8. A method in accordancewith claim 7, wherein said Type I IFN is IFNβ.
 9. A method in accordancewith claim 1, wherein said IFNAR is the beta subunit of the humaninterferon α/β receptor (IFNAR2).
 10. A method in accordance with claim1, wherein said native human IFNAR polypeptide chain of e) is theextracellular domain of a native human IFNAR polypeptide chain.
 11. Amethod in accordance with claim 3, wherein said native human IFNARpolypeptide chain of e) is the extracellular domain of a native humanIFNAR polypeptide chain.
 12. A method for potentiating the biologicaleffects of Type I interferon (IFN), comprising: administering to apatient in need of Type I IFN therapy a subunit of the human interferonα/β receptor (IFNAR) which is capable of binding to the Type I IFN to bepotentiated, in an amount effective to provide such IFN therapy, whereinsaid IFNAR has a sequence consisting essentially of the sequence of a) anative human IFNAR polypeptide chain; b) a fragment of a) which hasIFNAR receptor agonist or antagonist activity; c) a variant of a) or b)which has at least 70% sequence identity with a) or b) and which hasIFNAR receptor agonist or antagonist activity; or d) a variant of a) orb) which is encoded by a DNA sequence which hybridizes to the complementof the native DNA sequence encoding a) or b) under moderately stringentconditions and which has Type I IFN receptor agonist or antagonistactivity; or a salt or functional derivative of a), b), c), or d) whichhas IFNAR receptor agonist or antagonist activity.
 13. An isolatedmolecule comprising a complex of a Type I interferon (IFN) and a subunitof the human interferon α/β receptor (IFNAR) which is capable of bindingto the Type I IFN of the complex, in which said Type I IFN is bound tosaid IFNAR by a covalent bond or a peptide bond, wherein said Type I IFNhas a sequence consisting essentially of the sequence of a) a nativeType I IFN; b) a fragment of a) which has Type I IFN receptor agonist orantagonist activity; c) a variant of a) or b) which has at least 70%sequence identity with a) or b) and which has Type I IFN receptoragonist or antagonist activity; or d) a variant of a) or b) which isencoded by a DNA sequence which hybridizes to the complement of thenative DNA sequence encoding a) or b) under moderately stringentconditions and which has Type I IFN receptor agonist or antagonistactivity; or a functional derivative of a), b), c), or d) which has TypeI IFN receptor agonist or antagonist activity; and wherein said IFNARhas a sequence consisting essentially of the sequence of e) a nativehuman IFNAR polypeptide chain; f) a fragment of e) which has IFNARbiological activity; g) a variant of e) or f) which has at least 70%sequence identity with e) or f) and which has IFNAR biological activity;or h) a variant of e) or f) which is encoded by a DNA sequence whichhybridizes to the complement of the native DNA sequence encoding e) orf) under moderately stringent conditions and which has IFNAR biologicalactivity; or a salt or functional derivative of e), f), g), or h) whichhas IFNAR biological activity.
 14. A molecule in accordance with claim13, wherein said Type I IFN is bound to said IFNAR by a covalent bond.15. A molecule in accordance with claim 13, wherein said Type I IFN isbound to said IFNAR by a peptide bond.
 16. A molecule in accordance withclaim 15, wherein said Type 1 IFN is linked to said IFNAR by means of apeptide linker.
 17. A molecule in accordance with claim 16, wherein saidpeptide linker is (GGGGS)n (SEQ ID NO:1) wherein n=1-5.
 18. A moleculein accordance with claim 13, wherein said Type I IFN is an IFNα, anIFNβ, or an IFNω.
 19. A molecule in accordance with claim 18, whereinsaid Type I IFN is IFNβ.
 20. A molecule in accordance with claim 13,wherein said IFNAR is the beta subunit of the human interferon α/βreceptor (IFNAR2).
 21. A molecule in accordance with claim 13, whereinsaid native human IFNAR polypeptide chain of e) is the extracellulardomain of a native human IFNAR polypeptide chain.
 22. A DNA encoding afusion protein which is a molecule in accordance with claim
 15. 23. Ahost cell transformed with a vector carrying a DNA in accordance withclaim 22 in a manner which permits expression of said fusion protein.24. A method of making a fusion protein comprising culturing a host cellin accordance with claim 23 and recovering the fusion protein expressedthereby.
 25. A method for improving the shelf life of Type I interferon,comprising storing said interferon in the form of a complex inaccordance with claim 10 or a complex in which said IFN is bound to saidIFNAR by a non-covalent bond in a pharmaceutically acceptableformulation.
 26. A method in accordance with claim 25, wherein thepharmaceutically acceptable formulation is non-acidic.
 27. Apharmaceutical composition consisting essentially of a pharmaceuticallyacceptable carrier and a complex of a Type I interferon (IFN) and asubunit of the human interferon α/α receptor (IFNAR) which is capable ofbinding to the type I IFN of the complex, wherein said Type I IFN has asequence consisting essentially of the sequence of a) a native Type IIFN; b) a fragment of a) which has Type I IFN receptor agonist orantagonist activity; c) a variant of a) or b) which has at least 70%sequence identity with a) or b) and which has Type I IFN receptoragonist or antagonist activity; or d) a variant of a) or b) which isencoded by a DNA sequence which hybridizes to the complement of thenative DNA sequence encoding a) or b) under moderately stringentconditions and which has Type I IFN receptor agonist or antagonistactivity; or a salt or functional derivative of a), b), c), or d) whichhas Type I IFN receptor agonist or antagonist activity; and wherein saidIFNAR has a sequence consisting essentially of the sequence of e) anative human IFNAR polypeptide chain; f) a fragment of e) which hasIFNAR receptor agonist or antagonist activity; g) a variant of e) or f)which has at least 70% sequence identity with e) or f) and which hasIFNAR receptor agonist or antagonist activity; or h) a variant of e) orf) which is encoded by a DNA sequence which hybridizes to the complementof the native DNA sequence encoding e) or f) under moderately stringentconditions and which has IFNAR receptor agonist or antagonist activity;or a salt or functional derivative of e), f), g), or h) which has IFNARbiological activity.
 28. A pharmaceutical composition in accordance with claim 27, wherein said native human IFNAR polyeptide chain of e) isthe extracellular domain of a native human IFNAR polypeptide chain.